Abstract: Natural killer (NK) cells are innate lymphocytes that possess traits of adaptive immunity, such as memory formation. However, the molecular mechanisms by which NK cells persist to form memory cells are not well understood. Using single cell RNA sequencing, we identified two distinct effector NK cell (NKeff) populations following mouse cytomegalovirus (MCMV) infection. Ly6C? memory precursor (MP) NK cells displayed enhanced survival during the contraction phase in a Bcl2-dependent manner, and differentiated into Ly6C+ memory NK cells. Our studies show that a NK cell-intrinsic checkpoint is controlled by the transcription factor Fli1 which limits MP NK formation by regulating early effector NK cell fitness during viral infection. Building upon this discovery, we have designed methods and materials for modulating the molecular mechanisms that regulate memory cell fate in NK cells, such as genetically modified NK cells having a deletion in the gene for the transcription factor Fli1.